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Abstract: This study evaluated the effect of sealants

on enamel demineralization, focusing on physical

protection of the sealed enamel and fluoride protection

of the adjacent unsealed enamel. Occlusal fissures with

areas measuring 12 mm2 were delimited in 48 extracted

molars, randomly divided into 4 groups (n =12): 1) no

sealing; 2) sealing with a resin-modified glass-ionomer

(Vitremer™, 3M ESPE); 3) sealing with a fluoride-

releasing composite sealant (Clinpro Sealant™, 3M

ESPE); and 4) sealing with a non-fluoridated composite

sealant (Concise™, 3M ESPE). A 4-mm2 window was

outlined on the buccal enamel for analysis of fluoride

uptake. Following treatment, groups 2, 3 and 4 were

subjected to 5-days of pH-cycling, while group 1 was

kept in a moist environment at 37°C. Fluoride uptake

was assessed by dental biopsy, and the amount of

fluoride released to the cycling solutions was determined

by ion analysis. Enamel demineralization around the

sealants was evaluated by cross-sectional micro-

hardness analysis. Group 2 showed higher levels of

fluoride release ( P < 0.01) and uptake by enamel ( P <

0.05), and lower levels of demineralization ( P < 0.05)

than groups 3 and 4. Group 3 exhibited reduced

demineralization on unsealed enamel and provided

fluoride uptake in a distant enamel area, while group

4 did not. (J. Oral Sci. 47, 35-41, 2005)

Keywords: sealant; demineralization; fluoride.

IntroductionOver the last three decades, caries prevalence has

declined worldwide (1). However, the decline has not

occurred uniformly on all dental surfaces, and most new

carious lesions in children and adolescents are now located

on occlusal surfaces (2,3).

Non-invasive sealants have been recommended for

children and adolescents at risk of caries (4). Some studies

have suggested that the benefit provided by protecting

pits and fissures is based on good retention and the integrity

of the sealant material (5-8). However, since the retention

of the sealant is not permanent, this physical effect could

be enhanced if the material simultaneously released fluoride

(9,10).

Some reports comparing caries development around

composite sealants and ionomeric sealants have shown the

superiority of the resinous material (11-13) because of its

better retention (6,14). Other authors, however, found no

significant difference between these kinds of sealants

(6,15), or found a greater potential for caries inhibition in

ionomeric sealants (16). Thus, the development of

composite sealants containing fluoride and resin-modified

glass-ionomer sealants would combine the benefits of

enamel adhesion and fluoride-release in one material (17).

Previous studies have demonstrated that fluoride release

by glass-ionomer cement restorations ensures an

anticariogenic effect around the enamel (18,19) and on the

adjacent tooth (20,21). However, further research is still

necessary concerning fluoride release by occlusal sealants,

particularly its effect on fluoride uptake on buccal surfaces.

Journal of Oral Science, Vol. 47, No. 1, 35-41, 2005

Correspondence to Dr. Marcelo Henrique Napimoga, Cariology

Area, Faculty of Dentistry of Piracicaba, University of Campinas,

Piracicaba, SP, Brazil. Av. Limeira, 901 Caixa Postal 52, 13414-

903, Piracicaba, São Paulo, Brazil.

Tel: +55-19-3412-5321Fax: +55-19-3412-5218

E-mail: [email protected]

Fluoride-releasing capacity and cariostatic effect

provided by sealants

Maristela Maia Lobo, Giovana Daniela Pecharki, Cristiana Tengan,

Débora Dias da Silva, Elaine Pereira da Silva Tagliaferro

and Marcelo Henrique Napimoga

Cariology Area, Piracicaba School of Dentistry, University of Campinas,

São Paulo, Brazil

(Received 2 July 2004 and accepted 7 February 2005)

Original



36

Therefore, the aim of the present study was to evaluate

the cariostatic effect provided by three different occlusal

sealants; a resin-modified glass-ionomer (Vitremer™), a

fluoride-releasing composite sealant (Clinpro™Sealant)

and a non-fluoridated composite sealant (Concise™),focusing on the benefits of the physical barrier formed over

the sealed enamel, the fluoride protection for the unsealed

enamel adjacent to sealant, and the uptake of fluoride into

distant enamel.

Materials and MethodsEthical Aspects

This in vitro study using human teeth was deemed to

be ethical according to the Brazilian Guidelines (Resolution

196 of the National Health Council, 1996), and the protocol

was approved by the Institutional Ethics Committee of Piracicaba School of Dentistry, University of Campinas.

Three commercial restorative materials were tested: a

resin-modified glass-ionomer (RMGI), a fluoride-releasing

composite sealant (FRCS) and a non-fluoridated composite

sealant (NFCS). The authors have no connection with the

manufacturer of these products.

Tooth Selection and PreparationForty-eight impacted human third molars, extracted for

orthodontic reasons and free from macroscopic defects or

staining on the occlusal and buccal surfaces, were selected

for this study. After pumicing, their roots were sectioned

(Isomet, Buheler, Lake Bluff, IL, USA) and their crowns

were stored in a 0.1% aqueous thymol solution (pH 7.0)

at 4°C for at least a month (22). Then, teeth were randomly

divided into four groups (n = 12), according to the sealant

materials used (Fig. 1): Group 1; teeth were not sealed;

Group 2; teeth were sealed with a RMGI (Vitremer™, 3M

ESPE Dental Products, St. Paul, MN, USA); Group 3; teeth

were sealed with a FRCS (Clinpro™Sealant, 3M ESPE);

and Group 4; teeth were sealed with a NFCS (Concise, 3M

ESPE).

Using a digital caliper (Mahr Federal, Germany, UK),

the following areas were outlined on each tooth: an occlusal

area 3 mm long and 4 mm wide (12 mm2), with the main

occlusal fissure at its center, and a square window measuring

4 mm

2

, positioned on the buccal surface. The reasons fordelimitating the occlusal and buccal areas were,

respectively, to standardize the volume of sealant material

applied (3 mm3) and to create an area of distant enamel

for posterior analysis of fluoride uptake. The tooth crowns

were fixed with orthodontic wires to facilitate manipulation

of specimens. They were then identified and isolated with

an acid-resistant nail varnish (Colorama, São Paulo, Brazil),

excepting for the occlusal and buccal delimited areas.

Sealing Procedures

Sealants were applied according to the manufacturer’sinstructions, except for the RMGI samples, which were

treated with a 1:2 powder/liquid ratio (23). Tooth fissures

of groups 2, 3 and 4 were etched with 35% phosphoric acid

(3M ESPE) for 15 seconds and washed for the same time.

After slight air-drying, sealants were applied with a sharp

explorer under 10 × magnification (Meiji 2000, Meiji,

China) to avoid excessive spreading of the sealant and to

leave a bilateral rim 1 mm wide and 3 mm long of unsealed

adjacent enamel. Sealants were photopolymerized within

the recommended time (Optilux 500, Demetron/Kerr,

Danbury, CT, USA).

pH-Cycling RegimenGroup 1 was kept in a moist environment at 37°C, while

the sealed groups (2, 3 and 4) were subjected to a 5-day

pH-cycling model, simulating a high caries challenge,

essentially according to Featherstone et al. (24). Teeth

were individually immersed in 0.5 ml of a demineralizing

solution (De) (2 mM calcium, 2 mM phosphate in 0.075

M acetate buffer, pH 4.3) for 6 h, at 37°C. After this, they

were washed in distilled water for 10 s, dried with absorbent

paper and individually immersed in 0.5 ml of a

remineralizing solution (Re) (1.5 mM calcium, 0.9 mM

phosphate, 150 mM of KCl in 0.1 M Tris buffer, pH 7.0)

for 18 h, at 37°C (25). The solutions were changed daily.

At the end of each cycle, De and Re solutions were mixed

and stored at 4°C for posterior analysis of fluoride release.

Fluoride Uptake DeterminationImmediately after the pH-cycling phase, the occlusal

surfaces of all teeth (including Group 1) were protected

with wax, leaving exposed only the buccal enamel window

(4 mm2). Three layers of enamel were sequentially removed

from each specimen by immersion in 0.5 ml of an aqueous

solution of 0.5 M hydrochloric acid for 15, 30 and 60 sFig. 1 Schematic presentation of the study design.

Group 2 (RMGI)

Vitremer

n = 12

Group 3 (FRCS)

Clinpro sealant

n = 12

Group 4 (NFCS)

Concise

n = 12

5-Days pH cycling

Fluoride uptake analysis

Fluoride-release analysis

Group 1

No sealing

n = 1 2

Moist

environment,

37°C

Cross-sectional micro-hardness



37

under agitation. An equal volume of TISAB II (Orion

Research, Boston, MA, USA) pH 5.0, modified with 20

g NaOH/L, was added to each solution containing the

dissolved enamel layer (4,26).

Fluoride measurements were made in duplicate, usingan ion-specific electrode (Orion 96-09) and an ion-analyzer

(Orion EA-940) (Orion Research, Boston, MA, USA)

which had been calibrated previously with triplicate fluoride

standards (0.025 to 4.0 µg F/ml), prepared in 50% TISAB

II, containing 0.25 M/L HCl. Group 1 served as a control

for fluoride uptake, expressed per layer of removed enamel

rather than as a function of depth (27).

Analysis of Fluoride ReleaseThe amount of fluoride released by the sealants during

the pH-cycling regimen was analyzed daily, pooling thedemineralizing and remineralizing solutions after each

cycle. Measurement was made as described for fluoride

uptake, except different fluoride standards were used

(0.025 to 2.0 µg F/ml), in TISAB III. The cumulative

fluoride release in the De and Re solutions during the 5

days was used in the statistical analysis to estimate the

difference among the sealants.

Cross-sectional Micro-hardnessAfter fluoride analysis, teeth of groups 2, 3 and 4 were

tested for enamel cross-sectional micro-hardness (CSMH).

Teeth from group 1 were not tested, because there was no

sealing of the occlusal surface, and thus no reference of

sealed or unsealed enamel areas. The other teeth were

sectioned through the center of the occlusal sealed area,

with the cut being perpendicular to the fissure orientation.

One of the tooth halves was randomly selected and

embedded in epoxy resin, with the outer enamel surface

perpendicular to the resin surface. The samples were

serially polished with Al2O3 papers of 400, 600 and 1200-

grit (Carborundum, São Paulo, Brazil) and then cloth

polished with 1.0-µm diamond paste (Buehler Metadi,

Buheler, Lake Buff, ILL, USA). Cross-sectional micro-

hardness tests were performed using a Knoop diamond

under a 25-g load for 5 s (28,29) (FM-ARS, Future-Tech,

Tokyo, Japan). Two rows of five indentations each (at

depths 10, 20, 30, 40 and 50 µm from surface enamel) were

made at distances of 100 µm below and 100 µm above the

sealant margin, on sealed and unsealed enamel, respectively

(Fig. 2). The Knoop hardness units data (KHN) were

converted to mineral content (volume %) using the equation:

mineral content = 4.3 (√KHN ______

) + 11.3 according to

Featherstone et al. (28).

Statistical AnalysisFor fluoride uptake, the third layer was transformed

(square root) before applying ANOVA and Newman-Keuls

multiple-comparison tests (SAS/STAT Guide for personal

computers, SAS Institute, Cary, NC, USA, 2001) becausevariance was not homogeneous. The sum of fluoride

released in De and Re solutions during pH-cycling was

analyzed using the Krushkal-Wallis and Dunn test (α =

0.05). A multi-factor ANOVA with a split-split-plot design

was applied to the CSMH data to analyze the interactions

among the factors (sealants, position from the margin of

the sealant and depth from the enamel surface). A multiple

comparison Tukey test (α = 0.05) was chosen to check

differences in means within these factors.

ResultsThere was no significant difference in the level of

fluoride released during pH-cycling between FRCS and

NFCS (P < 0.01). However RMGI released more fluoride

than all the other groups (P < 0.01) (Table 1).

There were statistically significant differences in fluoride

uptake (Table 1) between sealants (P = 0.0009) for the first

enamel layer, with the RMGI group (Vitremer™) showing

the highest amount of incorporated fluoride compared

with FRCS, NFCS and the control. In the second and

third layers, however, these differences were not statistically

significant (P > 0.05).

Regarding the mineral content data, multi-factor ANOVA

(Table 2) revealed an important interaction among the

factors ‘sealant’ and ‘position from the sealant margin’ (P

= 0.00016). The Tukey test showed no statistically

significant differences between all the groups on the sealed

enamel (P > 0.05) (Fig. 3). However, on the unsealed

enamel area at depths 10 and 20 µm from the enamel

Fig. 2 Diagrammatic representation of the cross-sectional

micro-hardness assay. Two rows of five indentations

each (at depths 10, 20, 30, 40 and 50 µm from surface

enamel) were made at distances of 100 µm below(sealed enamel) and 100 µm above (unsealed enamel)

the sealant margin.



38

Table 1 Fluoride concentration in enamel (µg F/cm2) and sums of fluoride released during pH-cycling (µg F/ml) according to

groups, Means (SD; n = 12)

Table 2 Multi-factor analysis of variance for cross-sectional micro-hardness data



39

surface, group 2 showed less demineralization (P < 0.01)

than groups 3 and 4 (Fig. 3). At the other depths, group 2

and 3 did not differ from each other (P > 0.05), but were

statistically significantly different from group 4 (P < 0.01).

DiscussionA synergistic cariostatic effect would be expected to

occur as a function of integrating retention and fluoride-

releasing properties in sealant materials. Fluoride is a

world-wide recognized anticariogenic substance (18,30),

and its release from a dental material can be effectively

estimated in simulated caries procedures (31). In the

present study, the RMGI samples released the highest

amounts of fluoride, thus confirming previous results (32-

35). However, the FRCS group and the NFCS group

performed similarly. These results could be explained bythe differences in the composition between ionomeric and

resinous materials, resulting in subsequent differences in

fluoride releasing profiles (35). According to Asmussen

and Peutzfeldt (36), diffusion of water into the material is

necessary for the formation of hydrogen ions, which attack

the fluoride-containing glass particles, releasing fluoride.

Ionomeric materials are more permeable to water, and

this aspect enhances fluoride diffusion and release (36).

On the other hand, the matrix of resinous sealants is much

less hydrophilic, making fluoride release more difficult (37).

With regard to mineral content values, no deminerali-

zation could be demonstrated on sealed enamel in all

materials tested (Fig. 3). This result is consistent with

earlier reports (7,38,39) showing that the cariostatic benefit

of fissure sealing is provided by the formation of a physical

barrier that protects the fissure from plaque stagnation and

carious attack. However, in the present study, there was

demineralization on the enamel that was immediately

adjacent to the sealant and directly exposed to the pH-

cycling solutions (Fig. 3). As expected, the NFCS showed

no cariostatic effect at any of the depths evaluated for the

unsealed enamel. Méjare and Mjör (40) noted the same

results when comparing Concise™ and glass-ionomer as

fissure sealants.

On the other hand, resin-modified glass-ionomer showed

significantly reduced demineralization in the shallower

enamel depths (10 and 20 µm), where the acid attack

seemed to be stronger. The behavior of this material is

consistent with previous results in deciduous (39) and

permanent teeth (32). Reduction of demineralization

around resin-modified glass-ionomer restorations seems

to be a result of its fluoride-releasing capacity, since the

presence of fluoride in the fluid around the enamel crystals

during the caries challenge reduces demineralization and

enhances remineralization (31).

Fluoride uptake by buccal enamel confirmed the superior

fluoride-releasing capacity of the resin-modified glass-

ionomer, in relation to the other materials. The incorporation

of fluoride into the enamel adjacent to a fluoride-releasing

restorative material is a useful way to estimate its cariostaticeffect (41), since fluoride reduces enamel solubility (42).

The higher levels of fluoride uptake in the enamel adjacent

to glass-ionomers and resin-modified glass-ionomers can

be explained by the enamel mineral being continuously

lost after acid attacks and regained during the dynamics

of the caries process (18). Glass-ionomers and resin-

modified glass-ionomers release more fluoride and thus

provide a cariostatic effect when used for fissure sealing.

Their protective effect relies on the continuous fluoride

release from the remaining particles confined to fissure sites,

even in cases of significant loss of the material (6,9,40).These results for fluoride uptake on buccal enamel

confirm previous studies that demonstrated fluoride uptake

at areas located up to 7.5 mm from the restoration margins

(34) or on the adjacent tooth (43). Our results confirm that

fluoride released from resin-modified glass-ionomer is

capable of affecting not only enamel adjacent to the sealant

margin, but also enamel located distant from it.

In conclusion, the present study shows that the fluoride-

releasing capacity of resin-modified glass-ionomer provides

Fig. 3 Means (SD; n = 12) of mineral content (vol %) at

different distances from enamel surface (depth) for

each sealant. (A) Sealed enamel: 100 µm below themargin of the sealant; (B) Unsealed enamel: 100 µm

above the margin of the sealant.



40

cariostatic benefits in areas both close to the sealant and

distant from it. These properties would be especially

beneficial for patients with high caries risk.

AcknowledgmentsThe authors thank Professor Jaime Aparecido Cury,

who was responsible for the graduate laboratory classes

in “In vitro models in cariology”, and mentor of this study;

Livia Maria Andaló Tenuta for assistance in writing the

manuscript; Professor Glaucia Maria Bovi Ambrosano

for assistance with the statistical analysis and Ms. Mariza

de Jesus Carlos Soares and Mr. José Alfredo da Silva for

technical assistance.

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